Relative Gene Expression Analysis Research Articles

Analysis of Relative Gene Expression of some Brain-Pituitary-Gonad Axis Genes of the Ripe Wild and Captive Female Liza ramada

Analysis of Relative Gene Expression of some Brain-Pituitary-Gonad Axis Genes of the Ripe Wild and Captive Female Liza ramada

Zebrafish as a model for the study of wound healing in hyperglycemia

Background: For preclinical studies of diabetic wound therapy, zebrafish have many orthologous signalling pathways that play essential roles in wound healing and regeneration. Objectives: This study compares the expression of four essential wound-healing genes and caudal fin regeneration in hyperglycemic zebrafish (Danio rerio) to normal zebrafish. Methods: Hyperglycemia was induced by using the intraperitoneal injection method with 350 mg/BW streptozotocin on days one, three, and five. The regeneration of the zebrafish's caudal fin was observed on day 5 after amputation using a stereomicroscope, followed by sampling of the blastema to analyse gene expression. Caudal fin regeneration was analysed using Zeiss Zen 3.3 blue documentation; blood glucose levels were measured using a glucometer; and relative gene expression analysis of sonic hedgehog (shh), insulin-like growth factor 2a (igf2a), bone morphogenetic protein 2b (bmp2b), collagen 1a2 (col1a2) was performed by qRT-PCR method. Results: The glucose level in the hyperglycemia group was 203.2 mg/dL, and that in the normal group was 59.5 mg/dL. The authors found that igf2a, shh, bmp2b, and col1a2 expression were all downregulated in the hyperglycemia group and decreased in caudal fin regeneration. Conclusion: This novel adult zebrafish model of hyperglycemia significantly impairs gene expression and caudal fin regeneration.

Molecular characterization of Lernathropus kroyeri from intensive aquaculture and pathophysiology of infested sea bass

The copepod Lernathropus kroyeri constitutes one of the major parasites for the Mediterranean aquaculture, infesting the sea bass Dicentrarchus labrax causing thus disruptions of growth performance and occasionally mortalities. Despite the large spread and the high frequency of this parasite in mariculture farms of Eastern Mediterranean, L. kroyeri genetic profile from aquaculture as well as the pathophysiological response of D. labrax have not been studied so far. Keeping this in mind, in the present study we investigated the L. kroyeri infestation on D. labrax from two farms in Greece, examining both healthy and heavy parasitized individuals. Assays included histopathology, phylogenetic reconstruction of the parasite and physiological response of the fish by the means of antioxidant, inflammatory metabolic and stress related gene expression analysis at both mRNA and protein levels. Genetic analysis indicated that L. kroyeri composes a monophyletic group, highly phylogenetically distant from other congeneric groups. Heavy infested D. labrax witnessed a significantly increased immune response that further led to oxidative stress and metabolic alterations. Overall, our results demonstrate the, seasonally independent, high infestation of this parasitic copepods, which continue to affect Mediterranean intensive aquaculture systems.

The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 μg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Expression analysis of necroptosis related genes and lncRNAs in patients with pituitary neuroendocrine tumors

Necroptosis can either be the cause of tumorigenesis or it can impede its process. Recently, it has been proved that long non-coding RNAs (lncRNAs) have different crucial roles at cellular level, especially on cell death. Regarding the important role of necroptosis and lncRNAs in the pathogenesis of different cancers, especially pituitary adenomas (PAs), we assessed expression levels of two necroptosis related genes, namely TRADD and BIRC2, in addition to three related lncRNAs, namely FLVCR1-DT, MAGI2-AS3, and NEAT1 in PAs compared with adjacent normal tissues (ANTs). TRADD had no significant difference between two groups; however, BIRC2, FLVCR1-DT, MAGI2-AS3, and NEAT1 were upregulated in PAs compared to ANTs (Expression ratios [95% CI] = 2.3 [1.47–3.6], 2.13 [1.02–4.44], 3.01 [1.76–5.16] and 2.47 [1.37–4.45], respectively). When taking into account different types of PAs, significant upregulation of BIRC2, MAGI2-AS3 and NEAT1 was recorded in non-functioning PAs compared with corresponding ANTs (Expression ratios [95% CI] =1.9 [1.04–3.43], 2.69 [1.26–5.72] and 2.22 [0.98–5.01], respectively). Additionally, higher levels of BIRC2 were associated with higher flow of CSF (P value=0.048). Moreover, higher Knosp classified tumors had lower levels of BIRC2 (P value=0.001). Finally, lower levels of MAGI2-AS3 were associated with larger tumor size (P value=0.006). NEAT1 expression was correlated with FLVCR1-DT and TRADD. TRADD expression was correlated with FLVCR1-DT. Additional correlation was observed between expression of BIRC2 and MAGI2-AS3. In sum, this study provides evidence that dysregulated levels of studied genes could contribute to the pathogenesis of pituitary tumors.

Relative gene expression analysis of catalase in environmental RNA from Japanese medaka exposed to toxic chemicals

AbstractEnvironmental RNA (eRNA) has the potential as a non‐invasive tool for assessing the physiological status of macro‐organisms, yet the quantitative method for relative gene expression analysis is still underexplored. To bridge this gap, this study introduces a relative quantification method for eRNA, employing quantitative PCR (qPCR) to evaluate the expression of the antioxidant gene, catalase (cat), in the Japanese medaka Oryzias latipes exposed to two toxic chemicals for 96 h: chlorpyrifos (CPS) and carbamazepine. Our results showed that, in eRNA, genes frequently used as a reference for tissue or cells in conventional expression analysis correlated with each other. Also, one of them (elfa) exhibited less variability, showing its potential suitability as a reference gene in eRNA analyses. Additionally, cat expression levels in eRNA increased with increasing CPS concentrations, in a concentration‐response manner. These results suggest the promising potential of qPCR applications for eRNA in the monitoring of an organism's health and response to environmental changes. However, we observed disparities in gene expression levels of cat and beta‐actin (actb) between eRNA and whole fish body, indicating eRNA might have a biased origin. Further research is needed to uncover the origin of eRNA and determine the limitations and applicability domain of eRNA analysis.

Interferon-β deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV

Human immunodeficiency virus-1 (HIV-1) infects the central nervous system (CNS) and causes HIV-associated neurocognitive disorders (HAND) in about half of the population living with the virus despite combination anti-retroviral therapy (cART). HIV-1 activates the innate immune system, including the production of type 1 interferons (IFNs) α and β. Transgenic mice expressing HIV-1 envelope glycoprotein gp120 (HIVgp120tg) in the CNS develop memory impairment and share key neuropathological features and differential CNS gene expression with HIV patients, including the induction of IFN-stimulated genes (ISG). Here we show that knocking out IFNβ (IFNβKO) in HIVgp120tg and non-tg control mice impairs recognition and spatial memory, but does not affect anxiety-like behavior, locomotion, or vision. The neuropathology of HIVgp120tg mice is only moderately affected by the KO of IFNβ but in a sex-dependent fashion. Notably, in cerebral cortex of IFNβKO animals presynaptic terminals are reduced in males while neuronal dendrites are reduced in females. The IFNβKO results in the hippocampal CA1 region of both male and female HIVgp120tg mice in an ameliorated loss of neuronal presynaptic terminals but no protection of neuronal dendrites. Only female IFNβ-deficient HIVgp120tg mice display diminished microglial activation in cortex and hippocampus and increased astrocytosis in hippocampus compared to their IFNβ-expressing counterparts. RNA expression for some immune genes and ISGs is also affected in a sex-dependent way. The IFNβKO abrogates or diminishes the induction of MX1, DDX58, IRF7 and IRF9 in HIVgp120tg brains of both sexes. Expression analysis of neurotransmission related genes reveals an influence of IFNβ on multiple components with more pronounced changes in IFNβKO females. In contrast, the effects of IFNβKO on MAPK activities are independent of sex with pronounced reduction of active ERK1/2 but also of active p38 in the HIVgp120tg brain. In summary, our findings show that the absence of IFNβ impairs memory dependent behavior and modulates neuropathology in HIVgp120tg brains, indicating that its absence may facilitate development of HAND. Moreover, our data suggests that endogenous IFNβ plays a vital role in maintaining neuronal homeostasis and memory function.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

Unveiling the Cell Wall-Targeting Mechanisms and Multifaceted Virulence Modulation by a Eugenol Glycoconjugate against Aspergillus fumigatus: Insights from in vitro and in ovo Studies.

The primary objective of this study was to elucidate the putative cell wall-associated targets of compound 6i, a glycoconjugate of eugenol, in Aspergillus fumigatus, while also evaluating its toxicity and assessing histopathologic alterations in the liver, heart, and kidney of compound 6i-treated embryos using an in ovo model. To achieve this aim, compound 6i was synthesized, and a series of biochemical assays were performed to determine its impact on the fungal cell wall. Additionally, qRT-PCR and LC-MS/MS analyses were conducted to investigate changes in gene and protein expression profiles associated with melanin biosynthesis, conidiation, siderophore production, transcriptional regulation of β-glucan biosynthesis, and calcineurin activity in A. fumigatus. The experimental findings revealed that compound 6i exhibited notable antifungal activity against A. fumigatus by perturbing cell wall integrity, hindering ergosterol, glucan, and chitin biosynthesis, and inhibiting catalase production. Moreover, relative gene expression and proteomic analyses demonstrated that compound 6i exerted both down-regulatory and up-regulatory effects on several crucial genes and proteins involved in the aforementioned fungal processes. Furthermore, increased expression of oxidative stress-related proteins was observed in the presence of compound 6i. Notably, the glycoconjugate of eugenol did not elicit cytotoxicity in the liver, heart, and kidney of chick embryos. The current investigation elucidated the multifaceted mechanisms by which compound 6i exerts its antifungal effects against A. fumigatus, primarily through targeting cell wall components and signaling pathways. These findings underscore the potential of the eugenol glycoconjugate as a promising antifungal candidate, warranting further exploration and development for combating A. fumigatus infections.

The mechanism of melatonin promotion on cucumber seedling growth at different nitrogen levels

The supply level of exogenous nitrogen has a very important influence on the growth and development of cucumber. Insufficient or excessive nitrogen application will lead to metabolic disorders in the body and affect the formation of yield. Therefore, it is of great scientific and practical significance to explore the corresponding mitigation measures. Melatonin (MT) is a multi-regulatory molecule with pleiotropic effects on plant growth and development. A large number of studies have shown that the appropriate amount of melatonin supplementation is beneficial to plant growth and development by promoting root development, delaying leaf senescence, and improving fruit yield. However, the study of MT function combined with a detailed physiological analysis of nitrogen (N) absorption and metabolism in cucumber plants needs further strengthening. We performed hydroponic tests at different nitrogen levels to determine the metabolic processes associated with the enhanced tolerance to nitrogen in melatonin-treated cucumber (Cucucumis sativus L.) seedlings. Cucumber seedlings were sprayed with 100 μM melatonin or water and treated with different nitrogen in the growth chamber for 7 days. Nitrogen deficiency significantly inhibited seedling growth, and this growth inhibition was partially alleviated by melatonin. The expression analysis of related carbon and nitrogen genes showed that the genes whose expression was significantly altered by melatonin were mainly related to carbon (C) and nitrogen (N) metabolism. By enzyme activity and reactive oxygen content data analysis, melatonin-treated cucumber seedlings showed relatively stable carbon and nitrogen levels compared to untreated ones. In conclusion, MT can repair the impaired growth and development situation by regulating the nitrogen assimilation capacity and the balance between oxidation and oxidative metabolism and carbon metabolism in the cucumber under different nitrogen levels.

Kluyveromyces marxianus MTCC 1389 Augments Multi-stress Tolerance After Adaptation to Ethanol Stress.

During fermentation, yeast cells undergo various stresses that inhibit cell growth and ethanol production. Therefore, the ability to tolerate multiple stresses during fermentation is one of the important characteristics for yeast cells that can be used for commercial ethanol production. In the present study, we evaluated the multi-stress tolerance of parent and ethanol adapted Kluyveromyces marxianus MTCC1389 and their relative gene expression analysis. Multi-stress tolerance was confirmed by determining its cell viability, growth, and spot assay under oxidative, osmotic, thermal, and ethanol stress. During oxidative (0.8% H2O2) and osmotic stress (2M NaCl), there was significant cell viability of 90% and 50%, respectively, by adapted strain. On the other hand, under 45°C of thermal stress, the adapted strain was 80% viable while the parent strain was 60%. In gene expression analysis, the ethanol stress responsive gene ETP1 was significantly upregulated by 3.5 folds, the osmotic stress gene SLN1 was expressed by 3 folds, and the thermal stress responsive gene MSN2 was expressed by 7 folds. This study shows adaptive evolution for ethanol stress can develop other stress tolerances by changing relative gene expression of osmotic, oxidative, and thermal stress responsive genes.

SAT651 Human Adipose-derived Extracellular Vesicles (AdEVs) Increase Proinflammatory Markers In Renal And Endothelial Cells: An In Vitro Preliminary Study

Abstract Disclosure: C.A. Carvajal: None. M.P. Hernandez: None. P. Carrion: None. A. Tapia: None. J.A. Lopez: None. A. Vecchiola: None. C.E. Fardella: None. A. Sandoval-Borquez: None. During obesity, white adipose tissue (WAT), undergoes hypertrophic and hyperplastic changes by differentiation of preadipocytes into adipocytes, which increase the expression of PPARγ, FASN, FABP4, and adiponectin genes. These adipokines play a key role in obesity progression and in cell communication. WAT also causes a chronic inflammatory state that modifies the gene expression and secretome, including releasing of adipose-derived extracellular vesicles (AdEVs) that carry a specific cargo thought to modify different signaling pathways in target cells. Aim: To evaluate the effect of AdEVs in renal and endothelial cells and its inflammatory phenotype. Methods: Human SW872 preadipocytes and differentiated adipocytes were cultured and characterized by optical microscopy and Oil-Red-O stain. EVs from both preadipocytes and adipocytes were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, electron microscopy and w-blot. AdEVs (1x10^3) were added to renal HCD and endothelial EaHy926 cells for 24 hours. Expression of adipogenic differentiation genes in preadipocytes, adipocytes and their EVs, as well as the expression of inflammatory markers (IL-6 and IL-1B) were perfomed by RT-qPCR. Results: SW872 differentiated cells showed a classical adipocyte morphology and important accumulation of lipid-droplets. Isolated AdEVs have a donut shape morphology and size (50 - 150 nm) in accordance to the MISEV2018 guidelines, including also the EVs markers CD9 and Tsg101. A higher EVs concentration released from preadipocytes compared to adipocytes was observed (5.21x10^10 vs 0.44x10^10 particles/mL, p ≤0.05). The analysis of relative gene expression (2^-ΔΔCT), showed that adipocytes increase FAPB4 and adiponectin and decrease in PPARγ and FASN gene expression, with respect to preadipocytes (p ≤0.05). EVs from preadipocytes and adipocytes present similar relative expression of adiponectin, PPARγ and FASN than their parental cells. Finally, both renal or endothelial cells treated with AdEVs express higher levels of IL-6 and IL-1B that untreated cells (p≤ 0.05). Conclusion: EVs released from SW872 adipocytes and preadipocytes have similar gene expression as their parental cells. Treatment of both renal and endothelial cells with AdEVs showed an increase in inflammatory markers, as IL-6 and IL-1B. These preliminary results reveals novel insights about the impact of AdEVs in renal collecting duct and endothelial cells, which support novel roles of AdEVs -and its cargo- in human pathophysiology associated to obesity. Acknowledgements: This study was supported by grants ANID-FONDECYT 1212006, 3200646; CONICYT-FONDEQUIP EQM150023, SOCHED & CETREN UC. Presentation: Saturday, June 17, 2023

Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚβ2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma (CTCL) that is clinically characterized by pruritic erythroderma, generalized lymphadenopathy, and the presence of neoplastic, clonally related, CD4+ T cells in the skin, lymph nodes, and peripheral blood (PB). Despite advances in therapy, the prognosis remains poor, with a 5-year overall survival of 30% (Willemze, 2019). The malignant cells (Sézary cells) exhibit heterogeneous aberrant immunophenotypes most commonly involving the loss of CD2, CD7, and CD26 and the upregulation of immune checkpoint receptor programmed cell-death (PD-)1 (CD279) and the killer cell immunoglobulin-like MHC receptors (KIR) CD158k (Çetinözman et al., 2014Çetinözman F. Jansen P.M. Willemze R. Expression of programmed death-1 in skin biopsies of benign inflammatory vs. lymphomatous erythroderma.Br J Dermatol. 2014; 171: 499-504Crossref PubMed Scopus (24) Google Scholar; Di Raimondo et al., 2022Di Raimondo C. Rubio-Gonzalez B. Palmer J. Weisenburger D.D. Zain J. Wu X. et al.Expression of Immune Checkpoint Molecules PD1, PD-L1 and ICOS in Mycosis Fungoides and Sézary Syndrome: Association with Disease Stage and Clinical Outcome.Br J Dermatol. 2022; Crossref PubMed Scopus (3) Google Scholar; Najidh et al., 2021Najidh S. Tensen C.P. van der Sluijs-Gelling A.J. Teodosio C. Cats D. Mei H. et al.Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome.Blood. 2021; 138: 2539-2554Crossref PubMed Scopus (18) Google Scholar; Nguyen et al., 2017Nguyen G.H. Olson L.C. Magro C.M. Upregulation of inhibitory signaling receptor programmed death marker-1 (PD-1) in disease evolution from cutaneous lymphoid dyscrasias to mycosis fungoides and Sezary's syndrome.Ann Diagn Pathol. 2017; 28: 54-59Crossref PubMed Scopus (0) Google Scholar; Querfeld et al., 2018Querfeld C. Leung S. Myskowski P.L. Curran S.A. Goldman D.A. Heller G. et al.Primary T Cells from Cutaneous T-cell Lymphoma Skin Explants Display an Exhausted Immune Checkpoint Profile.Cancer Immunol Res. 2018; 6: 900-909Crossref PubMed Scopus (58) Google Scholar; Samimi et al., 2010Samimi S. Benoit B. Evans K. Wherry E.J. Showe L. Wysocka M. et al.Increased programmed death-1 expression on CD4+ T cells in cutaneous T-cell lymphoma: implications for immune suppression.Arch Dermatol. 2010; 146: 1382-1388Crossref PubMed Scopus (112) Google Scholar; Wada et al., 2011Wada D.A. Wilcox R.A. Harrington S.M. Kwon E.D. Ansell S.M. Comfere N.I. Programmed death 1 is expressed in cutaneous infiltrates of mycosis fungoides and Sézary syndrome.Am J Hematol. 2011; 86: 325-327Crossref PubMed Scopus (0) Google Scholar). The underlying mechanisms by which these aberrant immunophenotypes arise are not fully elucidated. An epigenetic mechanism responsible for deregulated gene expression and subsequent aberrant protein expression in CTCL involves the hyper-or hypomethylation of CpG sites in or near the promoter CpG island region.(Tensen et al., 2022Tensen C.P. Quint K.D. Vermeer M.H. Genetic and epigenetic insights into cutaneous T-cell lymphoma.Blood. 2022; 139: 15-33Crossref PubMed Scopus (16) Google Scholar). Using the Illumina 450K array platform, we previously showed that the genome-wide DNA methylation profiles of CD4+ T cells from SS patients were distinct from those of patients with benign erythroderma (BE) and healthy controls (HC) (van Doorn et al., 2016van Doorn R. Slieker R.C. Boonk S.E. Zoutman W.H. Goeman J.J. Bagot M. et al.Epigenomic Analysis of Sézary Syndrome Defines Patterns of Aberrant DNA Methylation and Identifies Diagnostic Markers.J Invest Dermatol. 2016; 136: 1876-1884Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Herein, we selected gene PDCD1 (encoding PD-1) which is of particular interest given its frequently aberrant protein expression levels in SS and with potentially important implications in pathogenesis and/or of diagnostic, therapeutic, and prognostic value. To this end, we performed combined analyses of DNA methylation, gene, and protein expression in a large cohort of SS patients. We first assessed the DNA methylation status of gene promoter locus PDCD1 using methylation-specific melting curve analysis (MS-MCA). Following approval from the ethical committee of Leiden University Medical Center, we collected PB samples from 38 patients with SS, 10 BE, and 4 HCs (Tables S1/S2). All individuals provided written informed consent. We performed peripheral blood mononuclear cell (PBMC) isolation, CD4+ T-cell enrichment by MACS cell depletion of non-CD4+ T cells, DNA isolation, DNA bisulfite conversion, and MS-MCA on bisulfite-converted DNA (detailed in Supplementary Methods). Qualitative assessment of the methylation data showed that the PDCD1 gene promoter was markedly hypomethylated in SS samples as compared to BE/HC samples (Figure 1a). The majority of SS samples (81%, 35/43) showed a partially (n=24), or completely (n=11) hypomethylated PDCD1 gene promoter. In contrast, the PDCD1 gene promoter was methylated in all BE/HC samples. We further examined the corresponding gene and protein expression levels. We applied relative gene expression analysis on CD4-enriched T lymphocytes from PB of 36 SS and 6 BE samples by using qPCR analysis of PDCD1. Stratification of SS patients into cases with hypomethylated (n=30) or predominantly methylated (n=6) PDCD1 gene promoters showed a significant inverse correlation between PDCD1 methylation status and mRNA levels. The average PDCD1 promoter mRNA levels among SS samples with hypomethylated gene promoters demonstrated a significantly higher expression when compared to methylated gene promoters from SS (p=0.0027) or BE (p=0.0232) samples (Figure 1b). The PDCD1 gene expression levels in the latter groups were comparable (p=0.3496). Next, we reviewed PD-1 protein expression in skin biopsies by immunohistochemistry (IHC). In 42 available SS skin biopsies–37 initial and 5 follow-up– membranous PD-1 expression was present in the majority of cases (86%, 36/42). For the latter, the presence of PD-1 protein expression on neoplastic CD4+ T cells in skin correlated with hypomethylated PDCD1 gene promoters (MS-MCA) on total circulating CD4+ T cells (92%, 33/36). Likewise, negative PD-1 protein expression correlated with a (predominantly) methylated gene promoter (83%, 5/6). PD-1 was hardly expressed in the two BE skin biopsies that were available for IHC staining. Interestingly, in two of five SS patients with paired diagnostic and follow-up skin biopsies, we observed reduced PD-1 expression in the follow-up compared to the initial biopsy. These changes in PD-1 expression corresponded with an altered methylation pattern of the PDCD1 gene promoter. This is illustrated for a SS case for which additional analyses were performed on the PD-1 protein expression on neoplastic CD4+ T cells in the skin (IHC) and PB (flow cytometry) (Figure 2, Figure S1 and S2). PD-1 is normally expressed on immune-competent hematopoietic cells and, following binding with one of its two natural ligands PD-L1/PD-L2, it attenuates antitumor immunity through the inhibition of T-cell activation. Conversely, unlike the majority of lymphoid malignancies (Kornepati et al., 2022Kornepati A.V.R. Vadlamudi R.K. Curiel T.J. Programmed death ligand 1 signals in cancer cells.Nat Rev Cancer. 2022; 22: 174-189Crossref PubMed Scopus (53) Google Scholar), tumor cell-intrinsic PD-1 expression is a common feature of Sézary cells whereas its ligands are often confined to small tumor-cell subsets (Di Raimondo et al., 2022Di Raimondo C. Rubio-Gonzalez B. Palmer J. Weisenburger D.D. Zain J. Wu X. et al.Expression of Immune Checkpoint Molecules PD1, PD-L1 and ICOS in Mycosis Fungoides and Sézary Syndrome: Association with Disease Stage and Clinical Outcome.Br J Dermatol. 2022; Crossref PubMed Scopus (3) Google Scholar). Similarly, PD-1 is generally not expressed by non-neoplastic T cells present in the lymphoma microenvironment. Herein, we report on the dynamic epigenetic regulation of PD-1 expression in SS through (hypo)methylation of the PDCD1 gene promoter. We found a consistent inverse correlation between PDCD1 gene promoter methylation, PDCD1 gene expression, and PD-1 membranous protein expression. Together with the reversible character of PDCD1 methylation as observed in follow-up samples, these data imply an active causative role rather than a correlative one, which is in line with the epigenetic regulation of PDCD1 as described in normal T cells undergoing maturation (Bally et al., 2026). Nonetheless, it is unlikely that the evident hypomethylation signature across SS samples is merely reflective of normal T-cell development as we did not observe this phenomenon in BE/HC samples. These data cannot rule out other potential molecular mechanisms involved. Furthermore, the precise functional consequences of upregulated PD-1 expression and its significance for the future application of immunotherapies in CTCL remain to be discovered. Accordingly, while therapeutic blockade of PD-1/PD-L1/L2 interactions has been successfully exploited in several (hematopoietic) malignancies, results in CTCL are variable and inconclusive thus far (Khodadoust et al., 2020Khodadoust M.S. Rook A.H. Porcu P. Foss F. Moskowitz A.J. Shustov A. et al.Pembrolizumab in Relapsed and Refractory Mycosis Fungoides and Sézary Syndrome: A Multicenter Phase II Study.J Clin Oncol. 2020; 38: 20-28Crossref PubMed Scopus (117) Google Scholar). These variable results illustrate that these therapies may not only lead to the activation of reactive T cells but also PD-1-expressing tumor cells. In this regard, the additive use of DNA methyltransferase inhibitors, that further stimulate hypomethylation, might impair the efficacy of PD-(L)1 inhibitors and should be used with caution. Moreover, in the pursuit of biomarkers to predict responsiveness to immunotherapies targeting the PD-1/PD-L1 axis in CTCL, several biomarkers have been explored including the assessment of PD-(L)1 expression (Di Raimondo, 2022), PD-L1 structural variants as genomic biomarkers (Beygi et al., 2021Beygi S. Fernandez-Pol S. Duran G. Wang E.B. Stehr H. Zehnder J.L. et al.Pembrolizumab in mycosis fungoides with PD-L1 structural variants.Blood Adv. 2021; 5: 771-774Crossref PubMed Scopus (13) Google Scholar) and a spatial biomarker (SpatialScore) reflecting the functional immune state of the tumor microenvironment (Philips et al., 2021). In line, the PDCD1 (hypo)methylation status might potentially serve as a predictive biomarker of PD-1 activation and its clinical importance should be further investigated in future studies. The authors declare no conflicts of interest. Bally et al., 2016Bally A.P. Austin J.W. Boss J.M. Genetic and Epigenetic Regulation of PD-1 Expression.J Immunol. 2016; 196: 2431-2437Crossref PubMed Google Scholar, Phillips et al., 2021Phillips D. Matusiak M. Gutierrez B.R. Bhate S.S. Barlow G.L. Jiang S. et al.Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma.Nat Commun. 2021; 12: 6726Crossref PubMed Scopus (34) Google Scholar, Willemze et al., 2019Willemze R. Cerroni L. Kempf W. Berti E. Facchetti F. Swerdlow S.H. et al.The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas.Blood. 2019; 133: 1703-1714Crossref PubMed Scopus (605) Google Scholar. The authors thank the patients and healthy controls who participated in the study. Data Availability Statement All relevant data analyzed during this study are included in the main text and the supplementary files. Author Contributions Conceptualization: SN, WHZ, CPT, and MHV; Investigation and analysis: SN, WHZ. Data analysis - histopathology: AMRS and RW. Supervision: CPT and MHV. Writing – original draft: SN; Writing – review and editing: all authors. All authors participated in discussions and data interpretation and approved the final version of the manuscript. Download .pptx (26.89 MB) Help with pptx files Download .pptx (.1 MB) Help with pptx files Download .xlsx (.02 MB) Help with xlsx files Download .xlsx (.01 MB) Help with xlsx files Download .docx (.02 MB) Help with docx files

MTOR signaling and endometrial receptivity in infertile women with intramural uterine leiomyomas

BackgroundReceptive endometrium is a restraining factor in the establishment of pregnancy in several estrogen-dependent gynecological disorders including uterine leiomyomas. Recently, data are beginning to accrue suggesting negative impact of non-cavity distorting intramural fibroids on molecular mediators of endometrial receptivity. The potential importance of the mammalian target of rapamycin (mTOR) signaling pathway has been suggested during embryo implantation. However, its exact role in fibroid-associated endometrium during the window of implantation is poorly defined. The objective of the study was to examine the expression and cellular distribution of key components of the mTOR signaling pathway during window of implantation in infertile women with non-cavity distorting intramural uterine leiomyomas (n = 24) as compared to fertile controls (n = 17). Relative gene expression analysis of mTOR, TSC1, and TSC2 was performed by real-time PCR. Immunohistochemistry was used to evaluate the expression of mTOR, phospho-mTOR (Serine 2448), TSC1, TSC2, phospho-TSC2 (Threonine 1462), and phospho-S6 ribosomal protein (Serines 235 and 236) and Ki67.ResultsIn comparison to fertile controls, statistically significant upregulation of mTOR (8.97-fold; p < 0.001) and downregulation of TSC2 mRNA (− 6.01-fold; p < 0.01) levels and cell-specific upregulation of proteins phospho-mTOR, phospho-TSC2, and phospho-S6 and downregulation of TSC1 and TSC2 were observed in infertile women. The ratio of p-mTOR/mTOR and p-TSC2/TSC2 was significantly higher in infertile women. Pearson’s correlation analysis revealed significant negative correlation between p-mTOR and TSC2 and positive correlation between p-mTOR and p-S6 in the infertile group. Increased Ki67 labelling index was observed in the glandular epithelium (GE) and stroma of endometrium from infertile women as compared to controls.ConclusionsLoss of TSC2 function and enhanced expression of activated mTOR and its downstream targets, observed in the infertile group, indicate heightened mTOR signaling which might contribute to impaired endometrial receptivity. Increased number of Ki67-positive nuclei suggests that enhanced mTOR signaling may help drive dysregulated proliferation of midsecretory endometrium leading to compromised fertility in women with non-cavity distorting intramural uterine leiomyomas. The present findings provide avenues for future investigation of mTOR pathway as a nonsurgical alternative for treatment of infertility in these patients.

Green cabbage supplementation influences the gene expression and fatty acid levels of adipose tissue in Chinese Wanxi White geese.

Dietary green cabbage was evaluated for its impact on fatty acid synthetic ability in different adipose tissues during fattening of Wanxi White geese. A total of 256 Wanxi White geese at their 70 days were randomly allocated into 4 groups with 4 replicates and fed 0%, 15%, 30%, and 45% fresh green cabbage (relative to dry matter), respectively, in each group. Adipose tissues (subcutaneous and abdominal fat), liver and blood were collected from 4 birds in each replicate at their 70, 80, 90, and 100 days for fatty acid composition, relative gene expression and serum lipid analysis. Two-way or three-way analysis of variance was used for analysis. The contents of palmitic acid (C16:0), palmitoleic acid (C16:1), linoleic acid (C18:2), and alpha-linolenic acid (C18:3) were feeding time dependently increased. The C16:0 and stearic acid (C18:0) were higher in abdominal fat, while C16:1, oleic acid (C18:1), and C18:2 were higher in subcutaneous fat. Geese fed 45% green cabbage exhibited highest level of C18:3. Geese fed green cabbage for 30 d exhibited higher level of C16:0 and C18:0 in abdominal fat, while geese fed 30% to 45% green cabbage exhibited higher C18:3 in subcutaneous fat. The expression of Acsl1 (p = 0.003) and Scd1 (p<0.0001) were decreased with green cabbage addition. Interaction between feeding time and adipose tissue affected elongation of long-chain fatty acids family member 6 (Elovl6), acyl-CoA synthetase longchain family member 1 (Acsl1), and stearoly-coA desaturase 1 (Scd1) gene expression levels (p = 0.013, p = 0.003, p = 0.005). Feeding time only affected serum lipid levels of free fatty acid and chylomicron. Higher contents of C16:0, C18:1, and C18:3 were associated with greater mRNA expression of Scd1 (p<0.0001), while higher level of C18:2 was associated with less mRNA expression of Scd1 (p<0.0001). Considering content of C18:2 and C18:3, 30% addition of green cabbage could be considered for fattening for 30 days in Wanxi White geese.

Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells

BackgroundHypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparativeDelta Ct, RefFinder, and the Livak method.ResultsGene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions.ConclusionsWe have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.

Effects of dietary yeast cell wall supplementation on growth performance, intestinal Campylobacter jejuni colonization, innate immune response, villus height, crypt depth, and slaughter characteristics of broiler chickens inoculated with Campylobacter jejuni at d 21

A study was conducted to assess the effects of a dietary yeast cell wall (YCW) with and without a Campylobacter jejuni (CJ) challenge. A total of 2,240-day-old Ross 708 males were randomly assigned within 8 treatments with a 4×2 factorial design, with 4 diets (negative control, positive control, YCW constant dose (400 g/ton), and YCW step-down dose (800/400/200 g/ton in the starter/grower/finisher diets, respectively) and with and without d 21 CJ oral gavage challenge at 5.2×107 CFU/mL. At d 0, 14, 28, and 41 body weights and feed consumption were measured to determine performance. At d 14, 28, and 42, 8 jejunal and ileal histology samples per treatment were collected for villi morphology measurements. At d 22 and 28 (1- and 7-days postinoculation), 24 ileal tissue samples per treatment were collected for relative gene expression analysis. At d 42, 24 cecal content samples per treatment were collected for CJ enumeration. Finally, on d 44, 96 birds per treatment were processed to determine carcass yield and 16 carcass rinses per treatment were collected to determine CJ prevalence after processing. Diet or inoculation did not impact broiler performance (P > 0.05). Limited differences were observed in intestinal morphology, and villus height and crypt depth were different only in the ileum at d 42 (P=0.0280 and P=0.0162, respectively). At d 1 postinoculation, differences between treatments inoculated with CJ and PBS were observed in the expression of avian beta defensin 10 (AvBD10), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) (P < 0.05). At d 7 postinoculation, expression of AvBD10, IL-1ß, and IL-10 was similar among all treatments (P > 0.05). At d 42, all birds, regardless the inoculation, had similar levels of CJ recovered from cecal contents (P > 0.05). After processing, carcass yield and CJ prevalence postchilling was similar in all treatments (P > 0.05). Overall, under the conditions of this study, the addition of YCW during a CJ challenge did not have an impact in growth performance, innate immune response, cecal colonization, carcass yield, or CJ prevalence after processing.

Identification of a survival associated gene trio in chemical induced breast cancer

Sporadic cases of breast cancer being more prevalent than the hereditary cases, can be largely attributed to environmental pollutants like polycyclic aromatic hydrocarbons (PAHs). The aim of the present study was to identify gene dysregulations and the associations in DMBA (a PAH) induced breast cancer. A breast cancer model was developed in Wistar rats (n = 40), using DMBA. Serum proteomics (2D electrophoresis and MALDI-TOF MS) followed by relative gene expression analysis in mammary glands were conducted to reach to the differential gene signatures. The candidate genes were subjected to survival analysis (by GEPIA2 and KM plotter) and infiltration analysis (by ImmuCellAI) for correlating gene expression with patient survival and immune cell infiltration respectively. Further, the regulatory network investigation (by Cytoscape) was performed to find out the transcription factors (TFs) and miRNAs of the concerned genes. A gene trio (ANXA5, MTG1, PPP2R5B), expressing differentially in early mammary carcinogenesis at 4 months (precancerous stage) till full-fledged cancerous stage (post 6 months) was identified. The altered gene expression was associated with less survival among breast cancer patients (n = 4019). The dysregulated expression also has a correlation with enhanced mammary infiltration of immune cells. Moreover, a regulatory network (comprising of 77 transcription factors and 50 miRNAs) involved in the regulation of candidate genes was also deciphered. The deregulated target genes can therefore be explored for reregulation via identified TFs and miRNAs, and survival thereby improved.